The proposed research involves defining the detailed molecular mechanisms by which thyrotropin releasing hormone (TRH) and thyroid hormones regulate the secretion (release of preformed) of thyrotropin (TSH). A cell culture system comprised of TSH-producing cells only has been developed which is responsive to physiological concentrations of triiodothyronine (T3) and thyroxine (T4) and to TRH. These cells are derived from mouse pituitary thyrotropic (TtT) tumors. We have shown that thryoid hormone regulation of TSH production is likely mediated via interaction with nuclear receptors in these cells. We have demonstrated the interrelationship between thyroid hormones and TRH in the control of TSH production and have shown regulation of the concentration of plasma membrane receptors for TRH, a mechanism whereby the thyrotrope may alter its sensitivity to TRH. The early effects of TRH in TtT cells will be characterized with particular regard to the role of cyclic nucleotides (cyclic AMP and cyclic GMP), calcium ion and anions in mediating this action and their role in the complex closed loop system involved in controlling TSH secretion. We will study the mechanism of regulation of TRH plasma membrane receptor concentration and its role in altering the TtT cell's sensitivity to TRH. We will determine the fate of the TRH-plasma membrane receptor complex and attempt to purify the receptor, and will localize any other physiologically relevant receptors for TRH. Lastly, we will characterize some extranuclear aspects of thyroid hormone action and metabolism with particular regard to possible effects at the plasma membrane, especially interactions with TRH, and define the cellular uptake and metabolism of T3 and T4. It is hoped that these studies will define the molecular mechanisms involved in regulation of TSH secretion, especially as it relates to the interaction of the major hormonal mediators, TRH and thyroid hormones.